Fluorescent Proteins and Their Use in Marine Biosciences,Biotechnology, and Proteomics

This review explores the field of fluorescent proteins (FPs) from the perspective of their marine origins and their applications in marine biotechnology and proteomics. FPs occur in hydrozoan, anthozoan, and copepodan species, and possibly in other metazoan niches as well. Many FPs exhibit unique photophysical and photochemical properties that are the source of exciting research opportunities and technological development. Wild-type FPs can be enhanced by mutagenetic modifications leading to variants with optimized fluorescence and new functionalities. Paradoxically, the benefits from ocean-derived FPs have been realized, first and foremost, for terrestrial organisms. In recent years, however, FPs have also made inroads into aquatic biosciences, primarily as genetically encoded fluorescent fusion tags for optical marking and tracking of proteins, organelles, and cells. Examples of FPs and applications summarized here testify to growing utilization of FP-based platform technologies in basic and applied biology of aquatic organisms. Hydra, sea squirt, zebrafish, striped bass, rainbow trout, salmonids, and various mussels are only a few of numerous instances where FPs have been used to address questions relevant to evolutionary and developmental research and aquaculture.     

You are what you eat: fatty acid profiles as a method to track the habitat movement of an insect

Tracking the movement of small organisms is of tremendous importance to understanding the ecology of populations, communities, and ecosystems. However, it remains one of the most difficult challenges facing the field of movement ecology. We developed an intrinsic marking technique for tracking small organisms using dietary fatty acid profiles as a biomarker as well as for clarifying source-sink dynamics between populations on a landscape level. Navel orangeworm moths (NOW), Amyelois transitella (Walker) (Lepidoptera: Pyralidae), raised on two different host plants with significantly different fatty acid profiles, were used to develop a model that distinguishes NOW based on their larval host plant. Wild NOW from both known and unknown host plants were used to validate the model. NOW fatty acid profiles showed striking similarities to the fatty acid profile of their host plant demonstrating that fatty acids can act as an intrinsic marking technique for quantifying the movement of small organisms. We anticipate that given sufficient spatial variation in dietary fatty acids, this technique will be useful in studying the movement of arthropods and other invertebrates particularly when addressing questions of source-sink dynamics.     

Studying the movement behavior of benthic macroinvertebrates with automated video tracking

Quantifying and understanding movement is critical for a wide range of questions in basic and applied ecology. Movement ecology is also fostered by technological advances that allow automated tracking for a wide range of animal species. However, for aquatic macroinvertebrates, such detailed methods do not yet exist. We developed a video tracking method for two different species of benthic macroinvertebrates, the crawling isopod Asellus aquaticus and the swimming fresh water amphipod Gammarus pulex. We tested the effects of different light sources and marking techniques on their movement behavior to establish the possibilities and limitations of the experimental protocol and to ensure that the basic handling of test specimens would not bias conclusions drawn from movement path analyses. To demonstrate the versatility of our method, we studied the influence of varying population densities on different movement parameters related to resting behavior, directionality, and step lengths. We found that our method allows studying species with different modes of dispersal and under different conditions. For example, we found that gammarids spend more time moving at higher population densities, while asellids rest more under similar conditions. At the same time, in response to higher densities, gammarids mostly decreased average step lengths, whereas asellids did not. Gammarids, however, were also more sensitive to general handling and marking than asellids. Our protocol for marking and video tracking can be easily adopted for other species of aquatic macroinvertebrates or testing conditions, for example, presence or absence of food sources, shelter, or predator cues. Nevertheless, limitations with regard to the marking protocol, material, and a species’ physical build need to be considered and tested before a wider application, particularly for swimming species. Data obtained with this approach can deepen the understanding of population dynamics on larger spatial scales and of the effects of different management strategies on a species’ dispersal potential.     

Effects of SmartWater,a fluorescent mark,on the dispersal,behavior, and biocontrol efficacy of Phytoseiulus persimilis

New marking methods for studying small biocontrol agents (especially predatory mites) are needed because many current techniques are expensive, ineffective or not applicable to small organisms. The objective of this study was to determine whether SmartWater, a liquid and permanent fluorescent dye, can be used to mark Phytoseiulus persimilis for experimentation without any deleterious effects on its dispersal, behavior, reproduction, and biocontrol efficacy. Our results show that there were no significant differences in movement, inter-plant dispersal, feeding behavior, survivability, and reproduction between marked P. persimilis and control individuals sprayed with water. We also found that the SmartWater mark lasted for the duration of the mites’ life, indicating strong durability over time. Marking efficacy may be reduced, due to a trade-off between batch marking efficacy and the possibility of drowning study organisms. However, we feel future research could improve liquid marking techniques that would reduce this risk. Overall, this study concludes that SmartWater could be a useful marking tool for predatory mites in both laboratory and field studies.

     

Automatic detection of fish and tracking of movement for ecology

1. Animal movement studies are conducted to monitor ecosystem health, understand ecological dynamics, and address management and conservation questions. In marine environments, traditional sampling and monitoring methods to measure animal movement are invasive, labor intensive, costly, and limited in the number of individuals that can be feasibly tracked. Automated detection and tracking of small‐scale movements of many animals through cameras are possible but are largely untested in field conditions, hampering applications to ecological questions.
2. Here, we aimed to test the ability of an automated object detection and object tracking pipeline to track small‐scale movement of many individuals in videos. We applied the pipeline to track fish movement in the field and characterize movement behavior. We automated the detection of a common fisheries species (yellowfin bream, Acanthopagrus australis) along a known movement passageway from underwater videos. We then tracked fish movement with three types of tracking algorithms (MOSSE, Seq‐NMS, and SiamMask) and evaluated their accuracy at characterizing movement.
3. We successfully detected yellowfin bream in a multispecies assemblage (F1 score =91%). At least 120 of the 169 individual bream present in videos were correctly identified and tracked. The accuracies among the three tracking architectures varied, with MOSSE and SiamMask achieving an accuracy of 78% and Seq‐NMS 84%.
4. By employing this integrated object detection and tracking pipeline, we demonstrated a noninvasive and reliable approach to studying fish behavior by tracking their movement under field conditions. These cost‐effective technologies provide a means for future studies to scale‐up the analysis of movement across many visual monitoring systems.

     

Three-Dimensional Tracking of Small Aquatic Organisms Using Fluorescent Nanoparticles

Tracking techniques are vital for the understanding of the biology and ecology of organisms. While such techniques have provided important information on the movement and migration of large animals, such as mammals and birds, scientific advances in understanding the individual behaviour and interactions of small (mm-scale) organisms have been hampered by constraints, such as the sizes of existing tracking devices, in existing tracking methods. By combining biology, chemistry and physics we here present a method that allows three-dimensional (3D) tracking of individual mm-sized aquatic organisms. The method is based on in-vivo labelling of the organisms with fluorescent nanoparticles, so-called quantum dots, and tracking of the organisms in 3D via the quantum-dot fluorescence using a synchronized multiple camera system. It allows for the efficient and simultaneous study of the behaviour of one as well as multiple individuals in large volumes of observation, thus enabling the study of behavioural interactions at the community scale. The method is non-perturbing – we demonstrate that the labelling is not affecting the behavioural response of the organisms – and is applicable over a wide range of taxa, including cladocerans as well as insects, suggesting that our methodological concept opens up for new research fields on individual behaviour of small animals. Hence, this offers opportunities to focus on important biological, ecological and behavioural questions never before possible to address.     

Persistence and growth of fecal Bacteroidales assessed by bromodeoxyuridine immunocapture

Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order Bacteroidales are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal Bacteroidales marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of Bacteroides vulgatus, the ability of Bacteroidales bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal Bacteroidales organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature, Bacteroidales genetic marker sequences persisted for at least 24 h and Bacteroidales fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal Bacteroidales human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific Bacteroidales sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.     

Rhodamine B as a collective marker for studying movements of small mammals

We present a new method of collective marking (rhodamine B) of small mammals that can be used under natural conditions. We examine the acceptance of marked baits, detection and persistence of the signal on the different kinds of hairs of two small species of rodents. Rhodamine B was ingested by captive animals and their hairs were dyed a fluorescent red coloration and observed over more than 150 days. Preliminary results obtained under field conditions tend to demonstrate that winter and summer movements could be detected by this technique. This new collective marking technique may be of great interest to study population turnover and movements of small mammals between habitat patches; it may represent an important method of assessing connectivity and permeability of landscapes by small mammals.     

The integrative effects of behavior and morphology on amphibian movement

Animal movement and dispersal are key factors in population dynamics and support complex ecosystem processes like cross‐boundary subsidies. Juvenile dispersal is an important mechanism for many species and often involves navigation in unfamiliar habitats. For species that metamorphose, such as amphibians, this transition from aquatic to terrestrial environments involves the growth and use of new morphological traits (e.g., legs). These traits strongly impact the fundamental ability of an organism to move in novel landscapes, but innate behaviors can regulate choices that result in the realized movements expressed. By assessing the integrative role of morphology and behavior, we can improve our understanding of juvenile movement, particularly in understudied organisms like amphibians. We assessed the roles of morphological (snout‐vent length and relative leg length) and performance (maximal jump distance) traits in shaping the free movement paths, measured through fluorescent powder tracking, in three anuran species, Pacific treefrog (Hyliola regilla), Western toad (Anaxyrus boreas), and Cascades frog (Rana cascadae). We standardized the measurement of these traits to compare the relative role of species' innate differences versus physical traits in shaping movement. Innate differences, captured by species identity, were the most significant factor influencing movement paths via total movement distance and path sinuosity. Relative leg length was an important contributor but significantly interacted with species identity. Maximal jump performance, which was significantly predicted by morphological traits, was not an important factor in movement behavior relative to species identity. The importance of species identity and associated behavioral differences in realized movement provide evidence for inherent species differences being central to the dispersal and movement of these species. This behavior may stem from niche partitioning of these sympatric species, yet it also calls into question assumptions generalizing anuran movement behavior. These species‐level effects are important in framing differences as past research is applied in management planning.     

被动式电子标签用于花鼠种群动态研究的可行性

在野生动物研究过程中经常需要对动物个体进行标记。传统的标记方法存在着标记物易脱落、可能影响被标记动物的生理和行为、难于进行个体识别等缺点。被动式电子标签(passive integrated transponder, PIT)是基于射频识别技术的电子标签,可用于体内标记,具有不易脱落消失且能够进行个体识别的优点。我们在凉水国家级自然保护区以PIT标签为标记物,采用标志重捕技术开展花鼠(Eutamias sibiricus)的种群动态研究,同时对应用PIT标签的可靠性进行了研究。2011年秋季至2012年春季共标记花鼠个体140只,其中86只被重捕至少1次。2011年秋季标记的72只个体中有38只在2012年春季被重捕。重捕的花鼠个体注射口愈合完好,体重无明显降低。结果表明PIT标签是一种安全可靠、识别迅速、数据准确、不易丢失、可循环使用的体内标记电子标签,适用于基于个体识别的小型哺乳动物生态学研究。     

Tracking the small with the smallest--using nanotechnology in tracking zooplankton

A major problem when studying behavior and migration of small organisms is that many of the questions addressed for larger animals are not possible to formulate due to constraints on tracking smaller animals. In aquatic ecosystems, this problem is particularly problematic for zoo- and phytoplankton, since tracking devices are too heavy to allow the organism to act naturally. However, recent advances in nanotechnology have made it possible to track individual animals and thereby to focus on important and urgent questions which previously have not been possible to address. Here we report on a novel approach to track movement and migratory behavior of millimeter sized aquatic animals, particularly Daphnia magna, using the commercially available nanometer sized fluorescent probes known as quantum dots. Experimental trials with and without quantum dots showed that they did not affect behavior, reproduction or mortality of the tested animals. Compared to previously used methods to label small animals, the nano-labeling method presented here offers considerable improvements including: 24 h fluorescence, studies in both light and darkness, much improved optical properties, potential to study large volumes and even track animals in semi-natural conditions. Hence, the suggested method, developed in close cooperation between biologists, chemists and physicists, offers new opportunities to routinely study zooplankton responses to light, food and predation, opening up advancements within research areas such as diel vertical/horizontal migration, partial migration and other differences in intra- and interspecific movements and migration.     

A novel method for identifying behavioural changes in animal movement data

A goal of animal movement analysis is to reveal behavioural mechanisms by which organisms utilize complex and variable environments. Statistical analysis of movement data is complicated by the fact that the data are multidimensional, autocorrelated and often marked by error and irregular measurement intervals or gappiness. Furthermore, movement data reflect behaviours that are themselves heterogeneous. Here, we model movement data as a subsampling of a continuous stochastic processes, and introduce the behavioural change point analysis (BCPA), a likelihood-based method that allows for the identification of significant structural changes. The BCPA is robust to gappiness and measurement error, computationally efficient, easy to implement and reveals structure that is otherwise difficult to discern. We apply the analysis to a GPS movement track of a northern fur seal ( Callorhinus ursinus ), revealing an unexpectedly complex diurnal behavioural profile, and demonstrate its robustness to the greater errors associated with the ARGOS tracking system. By informing empirical interpretation of movement data, we suggest that the BCPA can eventually motivate the development of mechanistic behavioural models.     

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.     

Persistence and Growth of Fecal Bacteroidales Assessed by Bromodeoxyuridine Immunocapture

Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order Bacteroidales are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal Bacteroidales marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of Bacteroides vulgatus, the ability of Bacteroidales bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal Bacteroidales organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature, Bacteroidales genetic marker sequences persisted for at least 24 h and Bacteroidales fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal Bacteroidales human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific Bacteroidales sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.     

A high‐resolution approach for the spatiotemporal analysis of forest canopy space using terrestrial laser scanning data

Forest canopies and tree crown structures are of high ecological importance. Measuring canopies and crowns by direct inventory methods is time‐consuming and of limited accuracy. High‐resolution inventory tools, in particular terrestrial laser scanning (TLS), is able to overcome these limitations and obtain three‐dimensional (3D) structural information about the canopy with a very high level of detail. The main objective of this study was to introduce a novel method to analyze spatiotemporal dynamics in canopy occupancy at the individual tree and local neighborhood level using high‐resolution 3D TLS data. For the analyses, a voxel grid approach was applied. The tree crowns were modeled through the combination of two approaches: the encasement of all crown points with a 3D α‐shape, which was then converted into a voxel grid, and the direct voxelization of the crown points. We show that canopy occupancy at individual tree level can be quantified as the crown volume occupied only by the respective tree or shared with neighboring trees. At the local neighborhood level, our method enables the precise determination of the extent of canopy space filling, the identification of tree–tree interactions, and the analysis of complementary space use. Using multitemporal TLS data recordings, this method allows the precise detection and quantification of changes in canopy occupancy through time. The method is applicable to a wide range of investigations in forest ecology research, including the study of tree diversity effects on forest productivity or growing space analyses for optimal tree growth. Due to the high accuracy of this novel method, it facilitates the precise analyses even of highly plastic individual tree crowns and, thus, the realistic representation of forest canopies. Moreover, our voxel grid framework is flexible enough to allow for the inclusion of further biotic and abiotic variables relevant to complex analyses of forest canopy dynamics.     

AeDNA: 水生生物eDNA数据库

环境 DNA (eDNA) 技术是一种生态和生物多样性监测和评价的新手段, 完整和准确的参考序列库是eDNA技术应用于水生生物多样性调查的基础。当前, 不同水生生物eDNA参考序列还存在诸多问题, 如不同类群使用的标记基因不同且资源较为分散, 部分参考序列分类不准确, 以及针对我国各类水体中水生生物eDNA参考序列不多等。针对上述问题, 研究构建了水生生物eDNA数据库(AeDNA, http://aedna.ihb.ac.cn/)。 AeDNA整合了DNA条形码和基因组两种类型参考序列。其中18S、28S、ITS、COΙ、12S、rbcL 等各类DNA条形码60余万条, 涉及2万余种鱼类、1万余种水生植物、1万余种底栖动物、1万余种浮游动物和1万余种浮游植物; 基因组包含线粒体、叶绿体等细胞器基因组6199个及万种鱼类基因组计划和万种原生生物基因组计划所产生的物种基因组。涉及的生境有江、河、湖、海、冰川和温泉等各类水环境, 尤其数据库构建团队贡献的6万余条参考序列, 具有我国丰富的各类水体生境信息。总体来说, AeDNA是一个数据量大、类群覆盖全、准确性高且具有我国水生生物特色的综合性eDNA参考序列库, 是水生态和水生生物多样性监测的重要基础资源。     

Tree Canopy Effects on Simulated Water Stress in Southern African Savannas

A coupled energy and water balance model is used to simulate the effects of large tree canopies on soil moisture and water stress across a series of sites spanning a regional moisture gradient in southern Africa. The model tracks evapotranspiration from five components of the land surface at each site—the tree canopy, the grass under and between tree canopies, and the bare soil under and between tree canopies. The soil moisture dynamics are simulated at daily time steps and driven by a stochastic model of storm arrivals and storm depth. Evapotranspiration is modeled using the Priestley-Taylor approach, with potential evapotranspiration scaled by soil moisture availability. The soil moisture under tree canopies is compared to the soil moisture between tree canopies, and differences in average annual soil moisture stress conditions are analyzed at each site. The spatial distribution of large trees has important consequences for small-scale soil moisture dynamics across the rainfall gradient. The results indicate that tree canopies serve to reduce soil moisture stress of under-canopy vegetation in the middle of the rainfall gradient. At the dry end of the rainfall gradient, the effect of tree canopies on soil moisture is dependent on the amount of rainfall received in a given growing season.     

核酸定量检测方法研究进展

核酸相关的研究和应用广泛存在于各个学科领域,与之相关的核酸定量检测技术越来越受到研究人员的重视。本文对目前实验室广泛采用的和近几年进展迅速的核酸定量方法(包括紫外分光光度法、荧光染料法、实时荧光定量PCR法、数字PCR法等)进行介绍,着重阐述其检测原理和优缺点,为研究人员今后进行相关研究提供参考。     

Linking habitat structure and orientation in an arboreal species Gehyra variegata (Gekkonidae)

Understanding how organisms move through landscapes is important for predicting the effects of landscape structure on the population dynamics and spatial distribution of organisms. Despite the accepted importance, the ability to orientate when moving is a poorly studied phenomenon. In this study we report on a translocation experiment in which we used fluorescent powder to study the ability of the arboreal gecko Gehyra variegata to orientate successfully between trees. The relocation experiment demonstrated the ability of translocated geckos to return to the tree of initial capture. Further, we investigated the set of rules geckos employ, when travelling through their structured habitat. Computer simulations relating capture-mark-recapture data to structural components of the habitat revealed that movement rules taking vision into account showed the best fit to the empirical data. The movement rule: "move randomly to one of the three next neighbouring trees that are visible" described the observed movement best. This movement rule connects all trees in the habitat and lowers the predation risk during movement.     

Tracking Image Correlation: Combining Single-Particle Tracking and Image Correlation

The interactions and coordination of biomolecules are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quantitative global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomolecules, usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization analysis along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle’s position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addition, the cross-correlation analysis shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments.